Hormones are biological messengers. They are synthesized by specific tissues (glands) and are secreted into the blood. The blood carries them to target cells where they act to alter the activities of the target cells.
Hormones are chemically diverse, and are generally categorized into three main groups: (1) small molecules derived from amino acids, for example thyroxine, (2) polypeptides or proteins, for example insulin and thyroid-stimulating hormone, and (3) molecules derived from cholesterol, for example steroids.
An important class of hormone is the thyroid hormones. Examples of thyroid hormones are thyroxine (T4), free thryoxine (FT4), triiodothyronine (T3) and free triiodothyronine (FT3). T4 and T3 enter cells and bind to intracellular receptors where they increase the metabolic capabilities of the cell by increasing mitochondria and mitochondrial enzymes. T4 and T3 are important in regulating a number of biological processes, including growth and development, carbohydrate metabolism, oxygen consumption, protein synthesis and fetal neurodevelopment. Synthesis of all circulating T4 and a small percentage of circulating T3 occurs on thyroglobulin molecules located within the thyroid. The bulk of the T3 present in the blood is produced enzymatically via monodeiodination of T4 by specific intracellular deiodinases—enzymes present in the follicular cells and the cells of target tissues [1]. In serum drawn from healthy human subjects, total T4 is present at about 60-fold higher concentration than total T3. T4 acts as a prohormone, as the reservoir for the production of T3, the active hormone. The metabolic activity associated with thyroid hormone (TH) is initiated by T3 binding to specific nuclear receptors within target cells. Thyroid hormone concentrations in blood are essential tests for the assessment of thyroid function.
Steroids make up another important class of hormones. Examples of steroid hormones include estrogens, progesterone and testosterone. Estrogen is the name of a group of hormones of which there are three principle forms, estrone, estradiol and estriol. Estrogens and progesterone cause the development of the female secondary sexual characteristics and develop and maintain the reproductive function. Testosterone develops and maintains the male secondary sex characteristics, promotes growth and formation of sperm. Steroids enter target cells and bind to intracellular receptors and then cause the production of mRNA coding for proteins that manifest the changes induced by steroids.
The accurate analysis and quantification of hormones is becoming more important. For example, estrogen and estrogen-like compounds are playing an ever-increasing role in today's society through hormone replacement therapy. Also, the analysis and quantification of estrogen and estrogen-like compounds helps in the management of estrogen-related diseases, like breast cancer. In addition, the accurate analysis and quantification of T4 and T3 is an issue recognized by those skilled in the art. The presence of circulating iodothyronine-binding autoantibodies that interfere with total T4 and T3 immunoassays (“IAs”) is a known phenomenon [2], [3], [4]. These autoantibodies may give falsely high, or falsely low values of thyroid hormone measurements depending on the assay separation method used, and are often in discordance with the clinical features [2], [3], [4]. Serum free T4 and T3 (FT4 and FT3) measurements are a way to compensate for such abnormal binding. However, technically, it is difficult to measure the free hormone concentrations since these are so low. It is easier to measure the total (free and protein-bound) thyroid hormone concentrations; total hormone concentrations are measured at nanomolar levels whereas free hormone concentrations are measured in the picomole range and to be valid, must be free from interference by the much higher total hormone concentrations.
Presently, the common methods of hormone analysis use immunoassay techniques. Table 1 lists the common hormones and the current methods for their analysis.
For example, estriol is analyzed by a radioimmunoassay utilizing radiolabelled antigen (iodine 125) in competition with unlabelled estriol in the sample, for a known amount of antibody. The assay is read using a gamma counter.
Androstenedione is analyzed using an enzyme immunoassay comprising horseradish peroxidase. Unlabeled antigen in the sample is in competition with enzyme labeled antigen for a fixed number of antibody binding sites. The assay is read using a microtitre plate enzyme immunoassay reader.
Several hormones are currently analyzed using a chemiluminescent immunoassay. For example, progesterone, testosterone, cortisol and T3 are analyzed using this method. The assay utilizes an assay-specific antibody-coated bead. The assay is read using a photon counter.
However, the current immunoassays are disadvantageous for the following reasons:                (1) Immunoassays are specific to one hormone, therefore every hormone must be analyzed separately.        (2) Numerous kits must be purchased and procedures must be learned for each hormone being analyzed.        (3) Various instruments to read the results from the immunoassays must be purchased.        
For example, the analysis of estriol and progesterone from a sample requires both a gamma counter and a photon counter.                (4) The kits for the assays can be expensive.        (5) The current immunoassays lack specificity and may show approximately 15 fold difference in results using kits from different manufacturers [5].        (6) The procedures involve many steps and can take a significant amount of time.        (7) In the case of a radioimmunoassay, precautions are necessary because of the radioisotopes involved.        
Immunoassays are notoriously unreliable with more and more literature published supporting their lack of specificity [6-13]. Table 2 shows the major differences reported by the College of American Pathologists program for proficiency testing of thyroid hormones that clearly illustrates the difference in specificity of the various antibodies used. For example, Table 2 shows mean results between different methods reported in the College of American Pathologists Proficiency Testing (CAP PT) Program can vary by a factor of approximately 2. Some factors such as pregnancy, estrogen therapy or genetic abnormalities in protein binding have also reportedly made immunoassay methods for T4 and T3 diagnostically unreliable [2], [3], [14], [15]. Currently serum total free T4 (FT4) and free T3 (FT3) concentrations are most commonly measured by immunoassay methods. Recently some reports of quantitative measurement of T4 and T3 by high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS/MS) were published [16-20]. All those methods required extraction, derivatization and even prior chromatographic separation that are very time-consuming [21], [22].
More recently, hormones have been analyzed and quantified by mass spectrometry. However, there are several disadvantages to these methods.
For example, a method of analyzing urinary testosterone and dihydrotestosterone glucuronides using electrospray tandem mass spectrometry has been described [23]. The method involves a complex system employing high performance liquid chromatography (HPLC) and a three-column two-switching valve. The shortcomings include the following: (i) the hormone glucuronides were analyzed, not the hormones, (ii) the method is applicable to urine only and (iii) only two analytes were analyzed simultaneously, (iv) the limit of detection (LOD) was 200 pg ml−1 for testosterone and the limit of quantification was 10 ug L−1 for dihydrotestosterone and (v) the method is complex.
Another publication discloses a method for the determination of estradiol in bovine plasma by an ion trap gas chromatography-tandem mass spectrometry technique [24]. The shortcomings include the following: (i) only one analyte was analyzed, (ii) 4 ml of plasma was required for the analysis of one analyte, (iii) the limit of detection was 5 pg ml−1, and (iv) derivation was required because the method employs gas chromatography.
A method for analysis of 17-hydroxyprogesterone by HPLC electrospray ionization tandem mass spectrometry from dried blood spots has also been described [25]. However, this method analyses only one analyte at a time, and requires liquid-liquid extraction, which is laborious and time consuming, with sample extraction alone taking 50 minutes to complete.
A gas chromatography mass spectrometry method to analyze the production rates of testosterone and dihydrosterone has been disclosed [26].
Finally, there is no known method of analyzing free thyroxine (FT4) or free triiodothyronine (FT3) by mass spectrometry. Most laboratories perform FT4 testing routinely employing the analogue (direct) immunoassay approach on one of the major clinical chemistry platforms. This approach is not universally accepted and has been the subject of criticism (29). There are frequent occasions when the validity of the FT4 result generated in this manner is questioned. For this reason a “reflex” testing for all direct FT4's<2.5th percentile is often done to diagnose hypothyroidism. These are sent out for FT4 measurements employing the current gold standard of equilibrium dialysis. This is also done for samples when the direct FT4 is >97.5th percentile and the TSH is normal. Approximately 50% of these FT4 send-outs have results within the normal range when measured by equilibrium dialysis and are therefore false positives by the direct FT4 method. However, the equilibrium dialysis procedures are time-consuming and expensive. Similarly, FT3 is also currently measured by immunoassay.
TABLE 1METHODS AND INSTRUMENTS FOR STEROIDAND THYROID HORMONES [1]PercentageAnalyteof UseInstrumentMethodAndrostenedione35%DSL solidEIA11-Deoxycortisol50%ICN Immuchem DARIADHEA Sulfate39%DPC ImmuliteECIAEstradiol16%Bayer ADVIA CentaurFIAEstriol,25%DSL liquidRIAunconjugatedEstriol, Total50%DPC Coat-a-CountRIA17-Hydroxy-51%DPC Coat-a-CountRIAprogesteroneProgesterone23%Bayer ADVIA CentaurCIATestosterone29%Bayer ADVIA CentaurCIATestosterone,65%DPC Coat-a-CountRIAFreeAldosterone76%DPC Coat-a-CountRIACortisol25%Bayer ADVIA CentaurCIACorticosteroneT329%Abbott AxsymFPIAT3, Free31%Bayer ADVIA CentaurCIAT430%Abbott AxsymFPIAT4, Free34%Abbott AxsymFPIARIA: RadioimmunoassayEIA: Enzyme Linked ImmunoassayFPIA: Fluorescence Polarization Immunoassay
TABLE 2Problems with Immunoassays: Data acquired fromCAP PT Program 2003Mean CAP ResultMean CAP Resultfor Methodfor MethodAnalyteGiving Lowest ValueGiving Highest ValueTriiodothyronine (ng/dL)108.5190.2364.8610.1Thyroxine (ug/dL)5.6410.091.643.658.7313.12